CD4+ T Cell Migration Assay

The method was used as described previously [23 (link)]. Briefly, the A_2AR-labeled, empty vector-labeled or blank ZM310 cells were grown on filters as mentioned above for 3 days. The CD4⁺ T cells were isolated from the spleen with Anti-Mouse CD4 Magnetic Particles (BD, #551539). Then, the medium of the upper chamber was changed to 100 μl 1640 medium containing CD4⁺ T cells (1 × 10⁶ cells/ml), and the insert was plated into a new well filled with RPMI 1640 (0.5% FBS). Twenty-four hours later, the migrating cells in the lower chamber were subjected to crystal violet staining and counted under a microscope (DM750, Leica).